1Biochemistry I Fall Term, 2004October 13 & 15, 2004Lecture 18: DNA Replication & RepairAssigned reading in Campbell: Chapter 9.1-9.6Key Terms:Leading and lagging strand synthesisExonucleolytic editingE. coli Pol I & Pol III holoenzymeNick translationDNA gyrase, DNA ligase, primase, & helicaseEukaryotic Pol α & Pol δReverse transcriptasePyrimidine dimersNucleotide excision repairProcessive polymerizationLinks:(I) Review Quiz on Lecture 18 concepts(I) DNA Replication Tutorial Flash animation of prokaryotic replication.(I) Enzymes & Proteins that act on DNA Handout is a worksheet to accompany this lecture.1. DNA Replication: an overview.Much of the complexity found in DNA replication (compared, say to transcription) derives fromthe requirements in all organisms for:• high accuracy in duplicating the primary genetic information;• high speed and processivity, so as to complete a copy of the genome in less than one cellgeneration (cell division time).Accuracy and speed cannot both be maximized; instead, each organism has evolved mechanismsto optimize these twin requirements. Moreover, it turns out that most of these mechanisms sharea common set of basic principles. Thus, the enzymes involved, the accessory proteins required,and the DNA structures they work on have been conserved in evolution from viruses, to bacteria,to yeast, to fruit flys, to humans.A. Replication Forks1. DNA polymerases can add nucleotides only to a pre-existing chain.2. All DNA (and RNA) polymerases synthesize chains from 5' --> 3'.3. DNA synthesis occurs at replication forks.2a) Synthesis is bidirectional from a replication origin (or origins).b) Replication forks are highly organized structures in cells (DNA synthesis"machines").B. Role of DNA Gyrase1. DNA gyrase introduces negative superhelical turns (endergonic).2. The reaction is coupled to ATP hydrolysis (net exergonic).C. Semidiscontinuous Replication1. The leading strand is synthesized continuously, 5' --> 3'.2. The lagging strand is synthesized in segments using RNA as primers (and as always,only in the 5' --> 3' direction).D. RNA primers1. Synthesized on the lagging strand template by DNA primase.2. Synthesized by RNA polymerase on the leading strand during replication initiation at theorigin.2. Enzymes of ReplicationSchematic diagram of DNA shown with several features that serve as the binding sites forenzymes and other proteins that function in replication and repair.A. DNA Polymerase I (Pol I): One enzyme - three active sites!1. Polymerization requires: a template, a primer strand, dNTPs, and Mg2+.2. The reaction produces: an extended primer and PPi.3. Pol I edits its own errors and those made by others (e.g. other enzymes, and other DNAdamaging events).a) 3' --> 5' exonuclease removes incorporation errors using a second active site(editing or proofreading). Products: dNMPs.b) 5' --> 3' exonuclease activity resides on a separate domain; it removes nucleotides3in lengths from 1 to 10 nucleotides.4. The 5' --> 3' exonuclease is essential for cell viability.a) this third active site removes damaged DNA starting from a "nick" (cleavage of thephosphodiester backbone, resulting in a 3'-OH and a 5'-phosphate).b) it also removes RNA from 5' ends of Okasaki fragments.c) "nick translation" results from the combined reactions of 5' --> 3' polymerizationand 5' --> 3' exonuclease activity.B. DNA Polymerase III (Pol III): The replicase machine of E. coli.1. Pol III can only fill in gaps in double strand DNA.2. Pol III has 10 subunits that assemble for replication.a) the ε subunit does the 3' --> 5' editing.b) the β subunit is the essential processivity factor (Campbell, Figs. 9.7 & 10).c) the α subunit has the polymerase active site.3. Pol III is present in only ~20 copies/cell.C. Helicases, Binding Proteins, and DNA Ligase.1. DnaB protein unwinds the parental helix (using ATP hydrolysis).2. The unwound single-stranded DNA is coated with SSB (ssDNA binding protein).3. DNA ligase repairs single-strand nicks, i.e. it catalyzes phosphodiester bond formationusing ATP (or NAD+) as energy donor. The "Okazaki fragments" that result from laggingstrand synthesis are joined by DNA ligase.3. Prokaryotic Replication MechanismsA. Escherichia coli1. Replication initiates at the oriC site.a) DnaA protein is required along with HU.b) About 45 bp of DNA are melted near the origin.c) DnaB and DnaC insert into the melted open complex to form a "preprimingcomplex" with the release of DnaC.4d) Assembly of accessory proteins follows.e) Pol III holoenzyme initiates synthesis on the leading and lagging strands.The two replication forks are closely associated during replication of the chromosome(See Fig. 9.10 in Campbell.). Recent studies show that this DNA replisome complex isfound at the center of the cell. This is also the location where specific cell divisionproteins assemble prior to the cell division event.Thus, the current picture of E. coli chromosome replication is one in which the DNAsynthesis and cell division "machines" are organized into coordinated replication"factories".2. Initiation of replication is coordinated with cell division; the mechanism(s) are unknown.3. Termination of Replication. D. Fidelity of Replication1. Balanced levels of dNTP's.2. Two-stage nucleotide incorporation.a) Watson-Crick base pairing (to the template base) and stacking (on the primerterminus).b) hydrolytic editing of errors (3' --> 5' exonuclease).The incorporation accuracy is about 1 error/106 nucleotides.3. Repair enzymes keep DNA under constant surveillance.The finished genome accuracy, i.e. including post-replication repair, approaches 1 error/109 basepairs. This is roughly one mutation per 1000 cells duplicated.4. Eukaryotic DNA ReplicationA. Eukaryotic DNA Polymerases.1. DNA Pol αααα replicates the lagging strand.2. DNA Pol δδδδ replicates the leading strand (with the PCNA processivity factor).3. Multiple replication origins are required.a) multiple origins allow S phase to be short in duration.b) clusters of replication "machines" are organized into many DNA synthesis"factories". This higher-order organization is very similar to that seen in E. coli,except that there are many more factories in eukaryotic cells during S phase.5B. Reverse Transcriptase (RT)1. Retroviruses have a single stranded RNA genome that is converted to double strandedDNA in three steps:a) RNA is the template for RT-catalyzed synthesis of an RNA-DNA duplex.b) The RNA is